Interaction of the Emerging Mycotoxins Beauvericin, Cyclopiazonic Acid, and Sterigmatocystin with Human Serum Albumin.

Beauvericin (BEA), cyclopiazonic acid (CPA), and sterigmatocystin (STC) are emerging mycotoxins. They appear as contaminants in food and animal feed, leading to economic losses and health risks. Human serum albumin (HSA) forms stable complexes with certain mycotoxins, including ochratoxins, alternariol, citrinin, and zearalenone. HSA binding can influence the toxicokinetics of xenobiotics, and albumin can also be considered and applied as a relatively cheap affinity protein. Therefore, we examined the potential interactions of BEA, CPA, and STC with HSA employing fluorescence spectroscopy, ultracentrifugation, ultrafiltration, and molecular modeling. Spectroscopic and ultracentrifugation studies demonstrated the formation of low-affinity BEA-HSA (Ka ≈ 103 L/mol) and moderately strong CPA-HSA and STC-HSA complexes (Ka ≈ 104 L/mol). In ultrafiltration experiments, CPA slightly displaced each site marker (warfarin, naproxen, and camptothecin) tested, while BEA and STC did not affect significantly the albumin binding of these drugs. Modeling studies suggest that CPA occupies Sudlow's site I, while STC binds to the Heme site (FA1) on HSA. Considering the interactions of CPA with the site markers, the CPA-HSA interaction may have toxicological importance.

Testing the extraction of 12 mycotoxins from aqueous solutions by insoluble beta-cyclodextrin bead polymer.

Mycotoxins are toxic metabolites of filamentous fungi; they are common contaminants in numerous foods and beverages. Cyclodextrins are ring-shaped oligosaccharides, which can form host-guest type complexes with certain mycotoxins. Insoluble beta-cyclodextrin bead polymer (BBP) extracted successfully some mycotoxins (e.g., alternariol and zearalenone) from aqueous solutions, including beverages. Therefore, in this study, we aimed to examine the ability of BBP to remove other 12 mycotoxins (including aflatoxin B1, aflatoxin M1, citrinin, dihydrocitrinone, cyclopiazonic acid, deoxynivalenol, ochratoxin A, patulin, sterigmatocystin, zearalanone, α-zearalanol, and ß-zearalanol) from different buffers (pH 3.0, 5.0, and 7.0). Our results showed that BBP can effectively extract citrinin, dihydrocitrinone, sterigmatocystin, zearalanone, α-zearalanol, and ß-zearalanol at each pH tested. However, for the removal of ochratoxin A, BBP was far the most effective at pH 3.0. Based on these observations, BBP may be a suitable mycotoxin binder to extract certain mycotoxins from aqueous solutions for decontamination and/or for analytical purposes.

The individual and combined effects of ochratoxin A with citrinin and their metabolites (ochratoxin B, ochratoxin C, and dihydrocitrinone) on 2D/3D cell cultures, and zebrafish embryo models.

Ochratoxin A and citrinin are nephrotoxic mycotoxins produced by Aspergillus, Penicillium, and/or Monascus species. The combined effects of ochratoxin A and citrinin have been examined in more studies; however, only limited data are available regarding the co-exposure to their metabolites. In this investigation, the individual toxic effects of ochratoxin A, ochratoxin B, ochratoxin C, citrinin, and dihydrocitrinone were tested as well as the combinations of ochratoxin A with the latter mycotoxins were examined on 2D and 3D cell cultures, and on zebrafish embryos. Our results demonstrate that even subtoxic concentrations of certain mycotoxins can increase the toxic impact of ochratoxin A. In addition, typically additive effects or synergism were observed as the combined effects of mycotoxins tested. These observations highlight that different cell lines (e.g. MDBK vs. MDCK), cell cultures (e.g. 2D vs. 3D), and models (e.g. in vitro vs. in vivo) can show different (sometimes opposite) impacts. Mycotoxin combinations considerably increased miR-731 levels in zebrafish embryos, which is an early marker of the toxicity on kidney development. These results underline that the co-exposure to mycotoxins (and/or mycotoxin metabolites) should be seriously considered, since even the barely toxic mycotoxins (or metabolites) in combinations can cause significant toxicity.

In Vitro Evaluation of the Individual and Combined Cytotoxic and Estrogenic Effects of Zearalenone, Its Reduced Metabolites, Alternariol, and Genistein.

Mycotoxins are toxic metabolites of filamentous fungi. Previous studies demonstrated the co-occurrence of Fusarium and Alternaria toxins, including zearalenone (ZEN), ZEN metabolites, and alternariol (AOH). These xenoestrogenic mycotoxins appear in soy-based meals and dietary supplements, resulting in the co-exposure to ZEN and AOH with the phytoestrogen genistein (GEN). In this study, the cytotoxic and estrogenic effects of ZEN, reduced ZEN metabolites, AOH, and GEN are examined to evaluate their individual and combined impacts. Our results demonstrate that reduced ZEN metabolites, AOH, and GEN can aggravate ZEN-induced toxicity; in addition, the compounds tested exerted mostly synergism or additive combined effects regarding cytotoxicity and/or estrogenicity. Therefore, these observations underline the importance and the considerable risk of mycotoxin co-exposure and the combined effects of mycoestrogens with phytoestrogens.

Interaction of silymarin components and their sulfate metabolites with human serum albumin and cytochrome P450 (2C9, 2C19, 2D6, and 3A4) enzymes.

Silymarin is a mixture of flavonolignans isolated from the fruit of milk thistle (Silybum marianum (L.) Gaertner). Milk thistle extract is the active ingredient of several medications and dietary supplements to treat liver injury/diseases. After the oral administration, flavonolignans are extensively biotransformed, resulting in the formation of sulfate and/or glucuronide metabolites. Previous studies demonstrated that silymarin components form stable complexes with serum albumin and can inhibit certain cytochrome P450 (CYP) enzymes. Nevertheless, in most of these investigations, silybin was tested; while no or only limited information is available regarding other silymarin components and metabolites. In this study, the interactions of five silymarin components (silybin A, silybin B, isosilybin A, silychristin, and 2,3-dehydrosilychristin) and their sulfate metabolites were examined with human serum albumin and CYP (2C9, 2C19, 2D6, and 3A4) enzymes. Our results demonstrate that each compound tested forms stable complexes with albumin, and certain silymarin components/metabolites can inhibit CYP enzymes. Most of the sulfate conjugates were less potent inhibitors of CYP enzymes, but 2,3-dehydrosilychristin-19-O-sulfate showed the strongest inhibitory effect on CYP3A4. Based on these observations, the simultaneous administration of high dose silymarin with medications should be carefully considered, because milk thistle flavonolignans and/or their sulfate metabolites may interfere with drug therapy.

Interactions of zearalanone, α-zearalanol, ß-zearalanol, zearalenone-14-sulfate, and zearalenone-14-glucoside with serum albumin.

The xenoestrogenic mycotoxin zearalenone is a Fusarium-derived food and feed contaminant. In mammals, the reduced (e.g., zearalanone, α-zearalanol, and ß-zearalanol) and conjugated (e.g., zearalenone-14-sulfate) metabolites of zearalenone are formed. Furthermore, filamentous fungi and plants are also able to convert zearalenone to conjugated derivatives, including zearalenone-14-sulfate and zearalenone-14-glucoside, respectively. Serum albumin is the dominant plasma protein in the circulation; it interacts with certain mycotoxins, affecting their toxicokinetics. In a previous investigation, we demonstrated the remarkable species differences regarding the albumin binding of zearalenone and zearalenols. In the current study, the interactions of zearalanone, α-zearalanol, ß-zearalanol, zearalenone-14-sulfate, and zearalenone-14-glucoside with human, bovine, porcine, and rat serum albumins were examined, employing fluorescence spectroscopy and affinity chromatography. Zearalanone, zearalanols, and zearalenone-14-sulfate form stable complexes with albumins tested (K = 9.3 × 103 to 8.5 × 105 L/mol), while the albumin binding of zearalenone-14-glucoside seems to be weak. Zearalenone-14-sulfate formed the most stable complexes with albumins examined. Considerable species differences were observed in the albumin binding of zearalenone metabolites, which may have a role in the interspecies differences regarding the toxicity of zearalenone.

Probing the Interactions of Ochratoxin B, Ochratoxin C, Patulin, Deoxynivalenol, and T-2 Toxin with Human Serum Albumin.

Ochratoxins, patulin, deoxynivalenol, and T-2 toxin are mycotoxins, and common contaminants in food and drinks. Human serum albumin (HSA) forms complexes with certain mycotoxins. Since HSA can affect the toxicokinetics of bound ligand molecules, the potential interactions of ochratoxin B (OTB), ochratoxin C (OTC), patulin, deoxynivalenol, and T-2 toxin with HSA were examined, employing spectroscopic (fluorescence, UV, and circular dichroism) and ultrafiltration techniques. Furthermore, the influence of albumin on the cytotoxicity of these xenobiotics was also evaluated in cell experiments. Fluorescence studies showed the formation of highly stable OTB-HSA and OTC-HSA complexes. Furthermore, fluorescence quenching and circular dichroism measurements suggest weak or no interaction of patulin, deoxynivalenol, and T-2 toxin with HSA. In ultrafiltration studies, OTB and OTC strongly displaced the Sudlow's site I ligand warfarin, while other mycotoxins tested did not affect either the albumin binding of warfarin or naproxen. The presence of HSA significantly decreased or even abolished the OTB- and OTC-induced cytotoxicity in cell experiments; however, the toxic impacts of patulin, deoxynivalenol, and T-2 toxin were not affected by HSA. In summary, the complex formation of OTB and OTC with albumin is relevant, whereas the interactions of patulin, deoxynivalenol, and T-2 toxin with HSA may have low toxicological importance.

Protective effects of beta-cyclodextrins vs. zearalenone-induced toxicity in HeLa cells and Tg(vtg1:mCherry) zebrafish embryos.

Zearalenone is a xenoestrogenic mycotoxin produced by Fusarium species. High exposure with zearalenone induces reproductive disorders worldwide. Cyclodextrins are ring-shaped host molecules built up from glucose units. The apolar cavity of cyclodextrins can entrap so-called guest molecules. The formation of highly stable host-guest type complexes with cyclodextrins can decrease the biological effect of the guest molecule. Therefore, cyclodextrins may be suitable to decrease the toxicity of some xenobiotics even after the exposure. In this study, the protective effect of beta-cyclodextrins against zearalenone-induced toxicity was investigated in HeLa cells and zebrafish embryos. Fluorescence spectroscopic studies demonstrated the formation of stable complexes of zearalenone with sulfobutyl-, methyl-, and succinyl-methyl-substituted beta-cyclodextrins at pH 7.4 (K = 1.4-4.7 × 104 L/mol). These chemically modified cyclodextrins considerably decreased or even abolished the zearalenone-induced loss of cell viability in HeLa cells and mortality in zebrafish embryos. Furthermore, the sublethal effects of zearalenone were also significantly alleviated by the co-treatment with beta-cyclodextrins. To test the estrogenic effect of the mycotoxin, a transgenic bioindicator zebrafish model (Tg(vtg1:mCherry)) was also applied. Our results suggest that the zearalenone-induced vitellogenin production is partly suppressed by the hepatotoxicity of zearalenone in zebrafish. This study demonstrates that the formation of stable zearalenone-cyclodextrin complexes can strongly decrease or even abolish the zearalenone-induced toxicity, both in vitro and in vivo. Therefore, cyclodextrins appear as promising new mycotoxin binders.

Cyclodextrins Can Entrap Zearalenone-14-Glucoside: Interaction of the Masked Mycotoxin with Cyclodextrins and Cyclodextrin Bead Polymer.

Zearalenone (ZEN) is a Fusarium-derived xenoestrogenic mycotoxin. In plants, zearalenone-14-O-ß-d-glucoside (Z14G) is the major conjugated metabolite of ZEN, and is a masked mycotoxin. Masked mycotoxins are plant-modified derivatives, which are not routinely screened in food and feed samples. Cyclodextrins (CDs) are cyclic oligosaccharides built up from D-glucopyranose units. CDs can form stable host-guest type complexes with lipophilic molecules (e.g., with some mycotoxins). In this study, the interaction of Z14G with native and chemically modified ß- and γ-CDs was examined employing fluorescence spectroscopy and molecular modeling. Furthermore, the removal of Z14G from aqueous solution by insoluble ß-CD bead polymer (BBP) was also tested. Our results demonstrate that Z14G forms the most stable complexes with γ-CDs under acidic and neutral conditions (K ≈ 103 L/mol). Among the CDs tested, randomly methylated γ-CD induced the highest increase in the fluorescence of Z14G (7.1-fold) and formed the most stable complexes with the mycotoxin (K = 2 × 103 L/mol). Furthermore, BBP considerably reduced the Z14G content of aqueous solution. Based on these observations, CD technology seems a promising tool to improve the fluorescence analytical detection of Z14G and to discover new mycotoxin binders which can also remove masked mycotoxins (e.g., Z14G).

Interaction of Dihydrocitrinone with Native and Chemically Modified Cyclodextrins.

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Aspergillus, Penicillium, and Monascus genera. It appears as a contaminant in grains, fruits, and spices. After oral exposure to CIT, its major urinary metabolite, dihydrocitrinone (DHC) is formed, which can be detected in human urine and blood samples. Cyclodextrins (CDs) are ring-shaped molecules built up from glucose units. CDs can form host-guest type complexes with several compounds, including mycotoxins. In this study, the complex formation of DHC with native and chemically modified beta- and gamma-cyclodextrins was tested at a wide pH range, employing steady-state fluorescence spectroscopic and modeling studies. The weakly acidic environment favors the formation of DHC-CD complexes. Among the CDs tested, the quaternary-ammonium-γ-cyclodextrin (QAGCD) formed the most stable complexes with DHC. However, the quaternary-ammonium-ß-cyclodextrin (QABCD) induced the strongest enhancement in the fluorescence signal of DHC. Our results show that some of the chemically modified CDs are able to form stable complexes with DHC (logK = 3.2-3.4) and the complex formation can produce even a 20-fold increase in the fluorescence signal of DHC. Considering the above-listed observations, CD technology may be a promising tool to increase the sensitivity of the fluorescence detection of DHC.

Interaction of the mycotoxin metabolite dihydrocitrinone with serum albumin.

Citrinin (CIT) is a nephrotoxic mycotoxin produced by Penicillium, Monascus, and Aspergillus species. CIT appears as a contaminant in cereals, cereal-based products, fruits, nuts, and spices. During the biotransformation of CIT, its major urinary metabolite dihydrocitrinone (DHC) is formed. Albumin interacts with several compounds (including mycotoxins) affecting their tissue distribution and elimination. CIT-albumin interaction is known; however, the complex formation of DHC with albumin has not been reported previously. In this study, we aimed to investigate the interaction of DHC with albumin, employing fluorescence spectroscopy, circular dichroism, and molecular modeling studies. Furthermore, species differences and thermodynamics of the interaction as well as the effects of albumin on the acute in vitro toxicity of DHC and CIT were also tested. Our main observations/conclusions are as follows: (1) Fluorescence signal of DHC is strongly enhanced by albumin. (2) Formation of DHC-albumin complexes is supported by both fluorescence spectroscopic and circular dichroism studies. (3) DHC forms similarly stable complexes with human albumin (K~105 L/mol) as CIT. (4) DHC-albumin interaction did not show significant species differences (tested with human, bovine, porcine, and rat albumins). (5) Based on modeling studies and investigations with site markers, DHC occupies the Heme binding site (subdomain IB) on human albumin. (6) The presence of albumin significantly decreased the acute in vitro cytotoxic effects of both DHC and CIT on MDCK cell line.

Interaction of 2'R-ochratoxin A with Serum Albumins: Binding Site, Effects of Site Markers, Thermodynamics, Species Differences of Albumin-binding, and Influence of Albumin on Its Toxicity in MDCK Cells.

Ochratoxin A (OTA) is a nephrotoxic mycotoxin. Roasting of OTA-contaminated coffee results in the formation of 2'R-ochratoxin A (2'R-OTA), which appears in the blood of coffee drinkers. Human serum albumin (HSA) binds 2'R-OTA (and OTA) with high affinity; therefore, albumin may influence the tissue uptake and elimination of ochratoxins. We aimed to investigate the binding site of 2'R-OTA (verses OTA) in HSA and the displacing effects of site markers to explore which molecules can interfere with its albumin-binding. Affinity of 2'R-OTA toward albumins from various species (human, bovine, porcine and rat) was tested to evaluate the interspecies differences regarding 2'R-OTA-albumin interaction. Thermodynamic studies were performed to give a deeper insight into the molecular background of the complex formation. Besides fluorescence spectroscopic and modeling studies, effects of HSA, and fetal bovine serum on the cytotoxicity of 2'R-OTA and OTA were tested in MDCK kidney cell line in order to demonstrate the influence of albumin-binding on the cellular uptake of ochratoxins. Site markers displaced more effectively 2'R-OTA than OTA from HSA. Fluorescence and binding constants of 2'R-OTA-albumin and OTA-albumin complexes showed different tendencies. Albumin significantly decreased the cytotoxicity of ochratoxins. 2'R-OTA, even at sub-toxic concentrations, increased the toxic action of OTA.

Interactions of zearalenone and its reduced metabolites α-zearalenol and ß-zearalenol with serum albumins: species differences, binding sites, and thermodynamics.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species. ZEN mainly appears in cereals and related foodstuffs, causing reproductive disorders in animals, due to its xenoestrogenic effects. The main reduced metabolites of ZEN are α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL). Similarly to ZEN, ZELs can also activate estrogen receptors; moreover, α-ZEL is the most potent endocrine disruptor among these three compounds. Serum albumin is the most abundant plasma protein in the circulation; it affects the tissue distribution and elimination of several drugs and xenobiotics. Although ZEN binds to albumin with high affinity, albumin-binding of α-ZEL and ß-ZEL has not been investigated. In this study, the complex formation of ZEN, α-ZEL, and ß-ZEL with human (HSA), bovine (BSA), porcine (PSA), and rat serum albumins (RSA) was investigated by fluorescence spectroscopy, affinity chromatography, thermodynamic studies, and molecular modeling. Our main observations are as follows: (1) ZEN binds with higher affinity to albumins than α-ZEL and ß-ZEL. (2) The low binding affinity of ß-ZEL toward albumin may result from its different binding position or binding site. (3) The binding constants of the mycotoxin-albumin complexes significantly vary with the species. (4) From the thermodynamic point of view, the formation of ZEN-HSA and ZEN-RSA complexes are similar, while the formation of ZEN-BSA and ZEN-PSA complexes are markedly different. These results suggest that the toxicological relevance of ZEN-albumin and ZEL-albumin interactions may also be species-dependent.

Interaction of Ochratoxin A and Its Thermal Degradation Product 2'R-Ochratoxin A with Human Serum Albumin.

Ochratoxin A (OTA) is a toxic secondary metabolite produced by several fungal species of the genus Penicillium and Aspergillus. 2′R-Ochratoxin A (2′R-OTA) is a thermal isomerization product of OTA formed during food processing at high temperatures. Both compounds are detectable in human blood in concentrations between 0.02 and 0.41 µg/L with 2′R-OTA being only detectable in the blood of coffee drinkers. Humans have approximately a fifty-fold higher exposure through food consumption to OTA than to 2′R-OTA. In human blood, however, the differences between the concentrations of the two compounds is, on average, only a factor of two. To understand these unexpectedly high 2′R-OTA concentrations found in human blood, the affinity of this compound to the most abundant protein in human blood the human serum albumin (HSA) was studied and compared to that of OTA, which has a well-known high binding affinity. Using fluorescence spectroscopy, equilibrium dialysis, circular dichroism (CD), high performance affinity chromatography (HPAC), and molecular modelling experiments, the affinities of OTA and 2′R-OTA to HSA were determined and compared with each other. For the affinity of HSA towards OTA, a logK of 7.0⁻7.6 was calculated, while for its thermally produced isomer 2′R-OTA, a lower, but still high, logK of 6.2⁻6.4 was determined. The data of all experiments showed consistently that OTA has a higher affinity to HSA than 2′R-OTA. Thus, differences in the affinity to HSA cannot explain the relatively high levels of 2′R-OTA found in human blood samples.

Removal of Zearalenone and Zearalenols from Aqueous Solutions Using Insoluble Beta-Cyclodextrin Bead Polymer.

Zearalenone (ZEN) is a Fusarium-derived mycotoxin, exerting xenoestrogenic effects in animals and humans. ZEN and its derivatives commonly occur in cereals and cereal-based products. During the biotransformation of ZEN, its reduced metabolites, α-zearalenol (α-ZEL) and ß-zearalenol (ß-ZEL), are formed; α-ZEL is even more toxic than the parent compound ZEN. Since previous studies demonstrated that ZEN and ZELs form stable complexes with ß-cyclodextrins, it is reasonable to hypothesize that cyclodextrin polymers may be suitable for mycotoxin removal from aqueous solutions. In this study, the extraction of ZEN and ZELs from water, buffers, and corn beer was investigated, employing insoluble ß-cyclodextrin bead polymer (BBP) as a mycotoxin-binder. Our results demonstrate that even relatively small amounts of BBP can strongly decrease the mycotoxin content of aqueous solutions (including beer). After the first application of BBP for mycotoxin binding, BBP could be completely reactivated through the elimination of ZEN from the cyclodextrin cavities by washing with a 50 v/v% ethanol-water mixture. Therefore, our study suggests that insoluble cyclodextrin polymers may be suitable tools in the future to deplete mycotoxins from contaminated drinks.